Many functions of the vascular endothelium are distributed heterogeneously among mature anatomic sites or vascular beds. Although this distribution is important for maintaining tissue- and organ-specific vascular functions, mechanisms responsible for endothelial heterogeneity are largely unknown. Functional heterogeneity may reflect primary inherent differences in endothelial cell populations, differences induced by interactions with non-endothelial cell or tissue components, or a combination of these factors. The primary goal of the proposed experiments is to investigate how endothelial functional heterogeneity arises throughout development, whether functional specificity can be acquired at relatively early stages of ontogeny, and the contributions of cell-autonomous differentiation in endothelial sublineages to these processes. Cytokine- and endotoxin-responsive expression of ICAM-1 and E-selectin will be investigated because these functions are heterogeneous in mature endothelium and can be conveniently analyzed experimentally. Recent results suggest that ICAM-1 expression is responsive beginning at or near the earliest stages of endothelial ontogeny while E-selectin expression does not become responsive until later stages. The specific objectives of this project are to define further the developmental transitions that endothelial lineage cells manifest during progression from non-responsive to responsive states and to investigate the cellular and molecular mechanisms involved in these transitions. Experiments will be performed using murine embryos, differentiated embryoid bodies derived from murine embryonic stem cells, and cultured embryo and embryoid body cells to exploit the advantages of each experimental preparation. In Specific Aim 1, temporal and spatial relationships between cohorts of cells with different cytokine- and endotoxin-stimulus sensitivities and ICAM-1/E-selectin response profiles will be analyzed to understand better how the combinatorial heterogeneity of inducible adhesion molecule expression is specified and maintained. In Specific Aim 2, the applicant plans to investigate the role of tumor necrosis factor-(* receptor expression and of non-endothelial cells on the acquisition of endothelial inducibility. In Specific Aim 3, the applicant will determine whether or not a component of endothelial differentiation is cell-autonomous or whether or not differentiation is entirely dependent on interactions with other cells or their products. These experiments should contribute new and useful scientific knowledge about differentiation and specialization within the vascular endothelial lineage and the functional implications of these processes in health and disease.